Myosin plays critical roles in many cellular processes, including cytokinesis, cell locomotion, and developmentally regulated changes in cell morphology. In the Dictyostelium system heavy chain phosphorylation plays a key role in controlling myosin redistribution during contractile events, but almost nothing is known regarding the cellular and molecular properties of the myosin heavy chain kinases (MHCKs) that participate in these events. Recently developed gene targeting technologies, combined with molecular genetic, cell biological, and biochemical methodologies will allow us to establish the relationship between in vitro MHCK activities that have been observed and the in vivo functions of these proteins in regulation of myosin localization. One of the best candidates for a Dictyostelium MHCK with important physiological roles is a 130 kDa kinase that was purified by Cote and Bukiejko. This kinase is present in growth phase amoebae, and increases in abundance during the phase of development corresponding to active cell migration. In vitro, phosphorylation of Dictyostelium myosin by this kinase strongly inhibits filament formation at physiological ionic strength. The in vitro target sites for this kinase have been mapped to three threonine residues in the tall portion of the myosin heavy chain. During my postdoctoral work I used site-directed mutagenesis to demonstrate that these three residues are critically involved in regulation of myosin localization in vivo. Together these results suggest that the 130 kDa MHCK may play a critical role in the regulation of myosin recruitment and assembly in vivo. The specific aims of this proposal focus on the molecular genetic and cell biological characterization of this 130 kDa MHCK. These aims will include: 1) isolation of the Dictyostelium gene that encodes this MHCK and determination of its nucleotide sequence, 2) use of the cloned gene to generate MHCK- cell lines via gene targeting, and establishment of cell lines that overexpress the active kinase, 3) analysis of these cells lines addressing motility, cytokinesis, chemotaxis, and developmental behavior to elucidate in vivo functions of the MHCK, with respect to myosin- mediated roles and with respect to possible roles mediated by targets other than myosin, and 4) molecular genetic manipulation of the MHCK gene to produce mutant forms of the kinase, with the goal of dissecting the functional domains of the protein involved in specific aspects of its in vivo function and mechanisms by which its activity is regulated. This research will help identify the cellular mechanisms that regulate myosin organization and assembly into subcellular structures during events such as cell division, chemotaxis, and cell differentiation. Insights into these events will have fundamental implications for a variety of important processes, ranging from regulation of cell growth and cell division, to metastatic properties of tumor cells and chemotactic behavior of leukocytes, to mechanisms regulating differentiation and development.